Project Summary/Abstract Neurons in the central nervous system neurons have a limited ability to survive and regenerate their axons following injury. Damage to axons is complicit in several CNS disorders including glaucoma, a major focus of this proposal. Interventions to protect remaining cells and induce axonal regeneration have produced promising, yet ultimately unsatisfying, results, falling short of recovery. To improve upon these results, it is imperative to determine why some neurons fare better than others. Retinal ganglion cells (RGCs), whose axons pass through the optic nerve, offer a tractable system to study these processes. Our lab identified that the progression of RGC degeneration following axonal damage by optic nerve crush (ONC) is dependent on cell type. Additionally, multiple other labs have observed cell-type specific changes in RGC morphology and function in glaucoma models. Moreover, axon regeneration was also cell-type restricted following two different interventions targeting the mTor pathway after ONC. These findings demonstrate that cell-intrinsic characteristics have a strong influence on neuron degeneration and axonal regeneration. In this study, I will use high throughput single-cell transcriptomic approaches to examine gene expression in an unbiased manner in RGC populations after axonal perturbations. I will apply this approach under three sets of conditions as strategies to identify potential targets for neuroprotection and axonal regeneration. First, I will determine how gene expression differences emerge between resilient and susceptible types after ONC, at stages before susceptible types are lost. This will reveal expression changes that correlate with resilience. Second, I will assess gene expression changes induced by three established interventions that act on different pathways and promote survival and/or axonal regeneration in distinct subsets of RGCs. This will reveal the changes occurring in ?rescued? types that correlate with their improved outcomes. Third, I will assess gene expression in two models of glaucoma with distinct disease progression to identify shared and distinct mechanisms of degeneration. I will use insights gained from expression analyses to target candidate genes in vivo using loss and gain-of-function methods and determine their effects on RGC survival and/or axonal regeneration. This has important implications for understanding the different ways degeneration progresses in neurons and has strong potential to identify novel therapeutic targets for glaucoma and other neurodegenerative disorders.